Costunolide inhibits osteoclast differentiation by suppressing c-Fos transcriptional activity.

Costunolide, a sesquiterpene lactone, exhibits anti-inflammatory and anti-oxidant properties and mediates apoptosis. However, its effects and mechanism of action in osteoclasts remain unknown. Herein, we found that costunolide significantly inhibited RANKL-induced BMM differentiation into osteoclasts in a dose-dependent manner without affecting cytotoxicity. Costunolide did not regulate the early signaling pathways of RANKL, including the mitogen-activated protein kinase and NF-κB pathways. However, costunolide suppressed nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression via inhibition of c-Fos transcriptional activity without affecting RANKL-induced c-Fos expression. The inhibitory effects of costunolide were rescued by overexpression of constitutively active (CA)-NFATc1. Taken together, our results suggest that costunolide inhibited RANKL-induced osteoclast differentiation by suppressing RANKL-mediated c-Fos transcriptional activity.

Inhibitory regulation of osteoclast differentiation by interleukin-3 via regulation of c-Fos and Id protein expression.

Interleukin-3 (IL-3) is produced under various pathological conditions and is thought to be involved in the pathogenesis of inflammatory diseases; however, its function in bone homeostasis under normal conditions or nature of the downstream molecular targets remains unknown. Here we examined the effect of IL-3 on osteoclast differentiation from mouse and human bone marrow-derived macrophages (BMMs). Although IL-3 can induce osteoclast differentiation of multiple myeloma bone marrow cells, IL-3 greatly inhibited osteoclast differentiation of human BMMs isolated from healthy donors. These inhibitory effects of IL-3 were only observed at early time points (days 0 and 1). IL-3 inhibited the expression of c-Fos and NFATc1 in BMMs treated with RANKL. However, IL-3-mediated inhibition of osteoclast differentiation was not completely reversed by ectopic expression of c-Fos or NFATc1. Importantly, IL-3 induced inhibitor of DNA binding/differentiation (Id)1 in hBMMs, while Id2 were sustained during osteoclast differentiation of mBMMs treated with IL-3. Ectopic expression of NFATc1 in Id2-deficient BMMs completely reversed the inhibitory effect of IL-3 on osteoclast differentiation. Furthermore, inflammation-induced bone erosion was markedly inhibited by IL-3 administration. Taken together, our results suggest that IL-3 plays an inhibitory role in osteoclast differentiation by regulating c-Fos and Ids, and also exerts anti-bone erosion effects.

In vitro and in vivo osteogenic activity of licochalcone A.

We investigated the in vitro and in vivo osteogenic activity of licochalcone A. At low concentrations, licochalcone A stimulated the differentiation of mouse pre-osteoblastic MC3T3-E1 subclone 4 (MC4) cells and enhanced the bone morphogenetic protein (BMP)-2-induced stimulation of mouse bi-potential mesenchymal precursor C2C12 cells to commit to the osteoblast differentiation pathway. This osteogenic activity of licochalcone A was accompanied by the activation of extracellular-signal regulated kinase (ERK). The involvement of ERK was confirmed in a pharmacologic inhibition study. Additionally, noggin (a BMP antagonist) inhibited the osteogenic activity of licochalcone A in C2C12 cells. Licochalcone A also enhanced the BMP-2-stimulated expression of various BMP mRNAs. This suggested that the osteogenic action of licochalcone A in C2C12 cells could be dependent on BMP signaling and/or expression. We then tested the in vivo osteogenic activity of licochalcone A in two independent animal models. Licochalcone A accelerated the rate of skeletal development in zebrafish and enhanced woven bone formation over the periosteum of mouse calvarial bones. In summary, licochalcone A induced osteoblast differentiation with ERK activation in both MC4 and C2C12 cells and it exhibited in vivo osteogenic activity in zebrafish skeletal development and mouse calvarial bone formation. The dual action of licochalcone A in stimulating bone formation and inhibiting bone resorption, as described in a previous study, might be beneficial in treating bone-related disorders.

GM-CSF regulates fusion of mononuclear osteoclasts into bone-resorbing osteoclasts by activating the Ras/ERK pathway.

Osteoclasts are multinucleated cells that are formed by the fusion of mononuclear osteoclasts, which is an essential process in bone resorption leading to bone remodeling. Herein we show that GM-CSF promoted the fusion of prefusion osteoclasts (pOCs). The expression of GM-CSF receptor-alpha was significantly up-regulated at the fusion stage of pOCs induced by RANKL. GM-CSF induced the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which was mediated by inducing NFATc1 via induction of c-Fos. The expression of c-Fos and NFATc1 was regulated by the ERK signaling pathway. Inhibition of ERK and NFATc1 suppressed the expression of DC-STAMP and led to the fusion inhibition of pOC. However, retrovirus-mediated expression of NFATc1 in pOCs rescued the defect in pOC fusion, despite the presence of U0126 and cyclosporin A. GM-CSF-stimulated pOCs had an intact actin ring and could resorb bone. Importantly, pOCs infected with constitutively active MEK adenovirus expressed c-Fos and NFATc1, followed by the binding of NFATc1 to the DC-STAMP promoter, which enables its transcription and expression. Constitutively active MEK-infected pOCs are able to resorb bone by undergoing cell-cell fusion. Taken together, our results demonstrated that GM-CSF induced fusion of pOCs to form multinucleated osteoclasts, making the osteoclast capable of bone resorption.

Physcion-8-O-beta-D-glucopyranoside enhances the commitment of mouse mesenchymal progenitors into osteoblasts and their differentiation: Possible involvement of signaling pathways to activate BMP gene expression.

Here, we show the involvement of signaling pathways to induce the gene expression of bone morphogenetic protein (BMP) in the osteogenic activity of physcion-8-O-beta-D-glucopyranoside (physcion-Glu); it stimulated osteoblast differentiation in mouse osteoblast MC3T3-E1 subclone 4 cells and induced BMP-2 gene expression and activation of Akt and ERK/MAP kinases. Physcion-Glu-induced BMP-2 expression and mineralization were attenuated by LY294002, an inhibitor of PI3K that lies upstream of Akt and MAP kinases, suggesting that physcion-Glu induces osteoblast differentiation via PI3K-Akt/MAP kinase signaling pathways, which play important roles in inducing BMP-2 gene expression. Physcion-Glu also enhanced BMP-2-induced commitment of mouse bi-potential mesenchymal precursor C2C12 cells into osteoblasts while inducing the transcription of several osteogenic BMP isoforms, such as BMP-2, -4, -7, and -9. Osteogenic synergy between BMP-2 and physcion-Glu was supported by the fact that noggin inhibited BMP-2 and physcion-Glu-induced alkaline phosphatase expression and activity. Considering that physcion-Glu induced Runx2 activity and the nuclear translocation of p-Smad, physcion-Glu could act by enhancing the BMP signaling pathway that induces Smad activation and translocation to activate Runx2. In conclusion, physcion-Glu could enhance the commitment of mesenchymal progenitors into osteoblasts and their differentiation by activating signaling pathways to induce BMP gene expression.

N-acetylcysteine stimulates osteoblastic differentiation of mouse calvarial cells.

Estrogen deficiency causes osteoporosis via increased generation of reactive oxygen species (ROS), and thus, antioxidants may prove to be the effective therapeutic candidates. We examined the effects of the antioxidant N-acetylcysteine (NAC) on osteoblastic differentiation in mouse calvarial cells. NAC (10-30 mM) enhanced alkaline phosphatase activity, mRNA expression of osteoblast differentiation-associated genes and mineralized nodule formation. It also increased expression of bone morphogenetic proteins-2, -4, and -7. The osteogenic activity of NAC was partially reduced by inhibition of glutathione synthesis. Since caffeic acid phenethyl ester did not stimulate osteoblast differentiation, it is unlikely that ROS scavenging activity of NAC is sufficient for osteogenic activity. We observed that NAC suppressed small GTPase RhoA activity and activation of RhoA by Pasteurella multocida toxin suppressed the osteogenic activity of NAC. These results suggest that NAC might exert its osteogenic activity via increased glutathione synthesis and inhibition of RhoA activation.

Proteasome inhibitors induce osteoclast survival by activating the Akt pathway.

Osteoclasts rapidly undergo spontaneous apoptosis when deprived of survival factors. Regulation of osteoclast survival is important to treat bone-related diseases, such as osteoporosis. In this study, we found that the proteasome inhibitors, MG132 and ALLN, significantly inhibited osteoclast apoptosis induced by etoposide, as well as under conditions of survival factor deprivation. MG132 and ALLN inhibited the release of cytochrome c from mitochondria into the cytosol in the absence of survival factors and suppressed the cleavage of pro-caspase-9 and -3 to its active forms induced by etoposide. In addition, MG132 and ALLN enhanced the phosphorylation of Akt and ERK in osteoclasts. However, MG132 and ALLN did not inhibit the cleavage of caspase-9 and -3 in the presence of the phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002, while the inhibitory effect of MG132 and ALLN were intact in presence of the MEK1/2 inhibitor, U0126. LY294002 inhibited the survival of osteoclasts induced by MG132 and ALLN. Taken together, our results have demonstrated that proteasome inhibitors suppressed osteoclast apoptosis under conditions of survival factors deprivation through activation of the PI-3K/Akt pathway.

AG490, a Jak2-specific inhibitor, induces osteoclast survival by activating the Akt and ERK signaling pathways.

Osteoclasts are multinucleated cells with the unique ability to resorb bone. Elevated activity of these cells under pathologic conditions leads to the progression of bone erosion that occurs in osteoporosis, periodontal disease, and rheumatoid arthritis. Thus, the regulation of osteoclast apoptosis is important for bone homeostasis. In this study, we examined the effects of the Janus tyrosine kinase 2 specific inhibitor AG490 on osteoclast apoptosis. We found that AG490 greatly inhibited osteoclast apoptosis. AG490 stimulated the phosphorylation of Akt and ERK. Adenovirus-mediated expression of dominant negative (DN)-Akt and DN-Ras in osteoclasts inhibited the survival of osteoclasts despite the presence of AG490. Cytochrome c release during osteoclast apoptosis was inhibited by AG490 treatment, but this effect was inhibited in the presence of LY294002 or U0126. AG490 suppressed the proapoptotic proteins Bad and Bim, which was inhibited in osteoclasts infected with DN-Akt and DN-Ras adenovirus. In addition, constitutively active MEK and myristoylated-Akt adenovirus suppressed the cleavage of pro-caspase-9 and -3 and inhibited osteoclast apoptosis induced by etoposide. Taken together, our results suggest that AG490 inhibited cytochrome c release into the cytosol at least partly by inhibiting the pro-apoptotic proteins Bad and Bim, which in turn suppressed caspase-9 and -3 activation, thereby inhibiting osteoclast apoptosis.

Methanol extracts of Stewartia koreana inhibit cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression by blocking NF-kappaB transactivation in LPS-activated RAW 264.7 cells.

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are involved in various pathophysiological processes such as inflammation and carcinogenesis. In a search for inhibitors of COX-2 and iNOS production we found that extracts of Stewartia koreana strongly inhibited NO and PGE2 production in LPS-treated macrophage RAW 264.7 cells. We have now shown that the mRNA and protein levels of iNOS and COX-2 are reduced by the Stewartia koreana extract (SKE). SKE inhibited expression of an NF-kappaB reporter gene in response to LPS, and gel mobility shift assays revealed that SKE reduced NF-kappaB DNA-binding activity. The extract also inhibited LPS-induced phosphorylation of IkappaB-alpha and nuclear translocation of p65. Administration of the extract reduced the symptoms of arthritis in a collagen-induced arthritic mouse model. These results indicate that Stewartia extracts contain potentially useful agents for preventing and treating inflammatory diseases.

Inhibitory effects of Stewartia koreana on osteoclast differentiation and bone resorption.

Osteoclasts are responsible for bone lysis in several bone diseases such as osteoporosis and rheumatoid arthritis. Natural products from plants have been invaluable source in discovery of compounds for new therapies. In this study, we screened plant products for potential application to therapy for bone loss using a primary osteoclastogenesis culture system and found that extract of Stewartia koreana (SKE) had a strong inhibitory effect on osteoclast formation. To gain molecular insights, we examined the effect of SKE on signaling pathways and transcription factors stimulated by the osteoclast differentiation factor RANKL. SKE suppressed the induction of c-Fos and NFATc1 by RANKL. However, SKE did not inhibit NF-kappaB activation by RANKL. Among the MAPKs stimulated by RANKL, SKE significantly reduced the activation of ERK and p38. Therefore, the anti-osteoclastogenic effect of SKE is likely to be elicited by interference with RANKL signaling to ERK and p38, which mediate the induction of c-Fos and subsequently that of NFATc1. Consistent with the in vitro effect on osteoclast differentiation, SKE showed a great inhibitory effect on in vivo bone loss in LPS-challenged mice. Taken together, we demonstrated that SKE has inhibitory effects on osteoclast differentiation in vitro and confirmed its in vivo efficacy in prevention of inflammatory bone loss.

Chloroform extract of deer antler inhibits osteoclast differentiation and bone resorption.

It has been reported that deer antler extract has anti-bone resorptive activity in vivo. However, little is known about the cellular and molecular mechanism of this effect. In this study, we investigated the effects of deer antler extracts on osteoclast differentiation and bone-resorption in vitro. Chloroform extract (CE-C) of deer antler inhibited osteoclast differentiation in mouse bone marrow cultures stimulated by receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). CE-C suppressed the activation of extracellular signal-regulated kinase (ERK), protein kinase B (PKB/Akt) and inhibitor of kappa B (I-kappaB) by RANKL in osteoclast precursor cells. It also inhibited the bone resorptive activity of differentiated osteoclasts that was accompanied by disruption of actin rings and induction of the apoptosis. These results demonstrate deer antler extract may be a useful remedy for the treatment of bone-resorption diseases such as osteoporosis.

Glycyrrhizae Radix Inhibits Osteoclast Differentiation by Inhibiting c-Fos-Dependent NFATc1 Expression.

Osteoporosis results from imbalance between new bone formation and bone resorption leading to bone loss and is especially troublesome for postmenopausal women who suffer from estrogen deficiency. The ability of new therapeutic agents to treat this bone disease with minimal side effects has been extensively reported on and is continuously being sought out by researchers in this field. Thus, the purpose of this study was to investigate a natural herb that was already being used as a new treatment for osteoporosis. Here we found that water extract of Glycyrrhizae radix (GR) inhibits receptor activator of nuclear factor-[Formula: see text]B ligand (RANKL)-induced osteoclast differentiation in a dose-dependent manner without causing cytotoxicity. The mRNA expression of c-Fos, nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), and osteoclast-associated receptor (OSCAR) was considerably inhibited by GR treatment. GR inhibited RANKL-mediated c-Fos and NFATc1 expression in a dose-dependent manner. GR inhibited the degradation of I-[Formula: see text]B in RANKL-stimulated BMMs. However, GR-mediated inhibition of osteoclast differentiation and osteoclast-specific gene expression, including NFATc1, was reversed by ectopic expression of c-Fos. Also, GR significantly inhibited osteoclast formation in mouse calvariae in the presence of IL-1 and prostaglandin E2 (PGE2). Taken together, these results suggest that GR inhibited osteoclast differentiation, raising the possibility that GR may serve as a useful drug for osteoporosis.

Tanshinone IIA inhibits osteoclast differentiation through down-regulation of c-Fos and NFATc1.

Bone is a dynamic tissue that is regulated by the activity of bone-resorbing osteoclasts and bone-forming osteoblasts. Excessive osteoclast formation causes diseases such as osteoporosis and rheumatoid arthritis. Natural substances may be useful as therapeutic drugs to prevent many diseases in humans because they avoid the many side effects of treatment with chemical compounds. Here we show that tanshinone IIA isolated from Salvia miltiorrhiza Bunge inhibits the receptor activator of NF-kappaB ligand (RANKL)-mediated osteoclast differentiation of osteoclast precursors. Tanshinone IIA suppressed the expression levels of c-Fos and NFATc1 induced by RANKL. However, retrovirus-mediated overexpression of c-Fos induced the expression of NFATc1 despite the presence of tanshinone IIA and reversed the inhibitory effect of tanshinone IIA on osteoclast differentiation. Also, the introduction of osteoclast precursors with the NFATc1 retrovirus led to osteoclast differentiation in the presence of tanshinone IIA. Our results suggest that tanshinone IIA may have a role as a therapeutic drug in the treatment of bone disease such as osteoporosis.

Elucidation of CPX-1 involvement in RANKL-induced osteoclastogenesis by a proteomics approach.

To identify proteins potentially involved in osteoclast differentiation, we conducted a proteomics-based analysis using the osteoclastogenesis model cell line RAW264.7. Total proteins from undifferentiated cells, committed pre-osteoclasts, and differentiated osteoclasts were resolved by two-dimensional gel electrophoresis. Protein spots showing differential expression levels were processed for peptide mass fingerprinting. Among them, we identified the metallocarboxypeptidase CPX-1, which was prominently increased in pre-osteoclasts and then decreased in mature osteoclasts. Results of reverse transcription polymerase chain reaction, Western blot, and confocal microscopy were in agreement with the proteomics data. Notably, the forced overexpression of CPX-1 led to the inhibition of osteoclast formation, but not pre-osteoclast generation. Therefore, the transient up-regulation pattern of CPX-1 expression may be important for the successful progression from pre-osteoclasts to mature osteoclasts.

Inhibition of osteoclast differentiation and bone resorption by tanshinone IIA isolated from Salvia miltiorrhiza Bunge.

Osteoclasts, multinuclear cells specialized for bone resorption, differentiate from the monocyte/macrophage lineage of hematopoietic cells. Intervention in osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone diseases involving osteoclasts. In this study, we found that tanshinone IIA, originating from Salvia miltiorrhiza Bunge, inhibited the differentiation of osteoclasts. Addition of tanshinone IIA to the osteoclast precursor culture caused a significant decrease in the level of calcitonin receptor, c-Src, and integrin beta3 mRNA, which are normally upregulated during the osteoclast differentiation dependent on RANKL (receptor activator of nuclear factor kappa B ligand). RANKL activated the ERK, Akt, and NF-kappaB signal transduction pathways in osteoclast precursor cells, and tanshinone IIA suppressed this activation. Tanshinone IIA also inhibited the bone resorptive activity of differentiated osteoclasts, which was accompanied with the disruption of the actin ring. Thus, tanshinone IIA has the potential to ameliorate bone-resorption diseases in vivo by reducing both the number and activity of osteoclasts.

Inhibition of osteoclast differentiation and bone resorption by a novel lysophosphatidylcholine derivative, SCOH.

Osteoclasts are multinucleated cells formed by multiple steps of cell differentiation from progenitor cells of hematopoietic origin. Intervention in osteoclast differentiation is considered as an effective therapeutic approach to the treatment for bone diseases involving osteoclasts. In this study, we found that the organic compound (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine (SCOH) inhibited osteoclast differentiation. The inhibitory effect of SCOH was observed in mouse bone marrow cell cultures supported either by coculturing with osteoblasts or by adding macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-kappaB signal transduction pathways, and SCOH suppressed this activation. SCOH also inhibited the bone resorptive activity of differentiated osteoclasts. It attenuated bone resorption, actin ring formation, and survival of mature osteoclasts. Reduced activation of Akt and NF-kappaB and decreased induction of XIAP were observed in mature osteoclasts treated with SCOH. Thus, this novel phosphatidylcholine derivative may be useful for treating bone-resorption diseases.

Lipid rafts are important for the association of RANK and TRAF6.

Rafts, cholesterol- and sphingolipid-rich membrane microdomains, have been shown to play an important role in immune cell activation. More recently rafts were implicated in the signal transduction by members of the TNF receptor (TNFR) family. In this study, we provide evidences that the raft microdomain has a crucial role in RANK (receptor activator of NF-kappaB) signaling. We found that the majority of the ectopically expressed RANK and substantial portion of endogenous TRAF2 and TRAF6 were detected in the low-density raft fractions. In addition, TRAF6 association with rafts was increased by RANKL stimulation. The disruption of rafts blocked the TRAF6 translocation by RANK ligand and impeded the interaction between RANK and TRAF6. Our observations demonstrate that proper RANK signaling requires the function of raft membrane microdomains.

A hybrid peptide derived from cecropin-A and magainin-2 inhibits osteoclast differentiation.

The adult skeleton is in a dynamic state, being continually broken down and reformed by the coordinated actions of osteoclasts and osteoblasts. Increased osteoclast activity may contribute to the development of osteoporosis. Therefore, the intervention of osteoclast-mediated bone resorption is considered as an effective therapeutic approach in the treatment of osteoporosis. In the course of searching for agents that inhibit osteoclast differentiation and activation, we found that a novel hybrid peptide P1 derived from cecropin-A and magainin-2 reduced osteoclast differentiation in various osteoclast culture systems. As this peptide had no cytotoxicity on various cultures of primary cells and established cell lines, its inhibitory effect on osteoclastogenesis was not due to general cytotoxicity. The effects of P1 on osteoclasts appear to be mediated through the inhibition of NF-kappaB and JNK activation induced by the osteoclastogenic cytokine, receptor activator of NF-kappaB ligand (RANKL). These results provide an evidence for the potential usefulness of P1 for the treatment of bone-resorbing diseases.

The anti-proliferative gene TIS21 is involved in osteoclast differentiation.

The remodeling process of bone is accompanied by complex changes in the expression levels of various genes. Several approaches have been employed to detect differentially-expressed genes in regard to osteoclast differentiation. In order to identify the genes that are involved in osteoclast differentiation, we used a cDNAarray-nylon membrane. Among 1,200 genes that showed a measurable signal, 19 genes were chosen for further study. Eleven genes were up-regulated; eight genes were downregulated. TIS21 was one of the up-regulated genes which were highly expressed in mature osteoclasts. To verify the cDNA microarray results, we carried out RT-PCR and real-time RT-PCR for the TIS21 gene. The TIS21 mRNA level was higher in differentiated-osteoclasts when compared to undifferentiated bone-marrow macrophages. Furthermore, the treatment with 1 mM of a TIS21 antisense oligonucleotide reduced the formation of osteoclasts from the bone-marrow-precursor cells by approximately 30%. These results provide evidence for the potential role of TIS21 in the differentiation of osteoclasts

Effect of interferon-γ on the fusion of mononuclear osteoclasts into bone-resorbing osteoclasts.

Osteoclasts are multinucleated cells that are formed by the fusion of pre-fusion osteoclasts (pOCs). The fusion of pOCs is known to be important for osteoclastic bone resorption. Here, we examined the effect of IFN-γ on the fusion of pOCs. IFN-γ greatly increased the fusion of pOCs in a dose-dependent manner. Furthermore, IFN-γ induced pOC fusion even in hydroxyapatite-coated plates used as a substitute for bone. The resorption area of pOCs stimulated with IFN-γ was significantly higher than that of the control cells. IFN-γ induced the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which is responsible for the fusion of pOCs. IFN-γ enhanced DC-STAMP expression in a dose-dependent manner. The mRNA expression of c-Fos and nuclear factor of activated T cells (NFAT) c1 was enhanced in the pOCs treated with IFN-γ. Taken together, these results provide a new insight into the novel role of IFN-γ on the fusion of pOCs.